Abstract

Cancer is one of the most fatal diseases in humans, accounting for 13 % of all deaths worldwide through the destruction of healthy tissue and organs by uncontrolled proliferation. The most life-threatening step during tumor progression is reached when cancer cells leave the primary tumor mass to invade the surrounding tissue and colonize distant organs. This process of metastasis includes dissemination throughout the body by entering the blood - or lymphatic system to reach distant organs and form secondary tumors. To infiltrate the surrounding tissue, single motile tumor cells leave the tumor mass by breaking down cell-cell contacts, in a process called epithelial to mesenchymal transition (EMT). EMT is a complex molecular and cellular program enabling epithelial cells to abandon their differentiated phenotype, including cell-cell adhesion and cell polarity, and to acquire mesenchymal features and invasive properties. One of the most prominent inducers of EMT is transforming growth factor β (TGFβ), which can suppress tumor formation by growth inhibition but also promotes tumor progression and metastasis in advanced tumors. To dissect the molecular mechanism underlying the complex process of EMT and understand the dual role of TGFβ during cancer progression we established different in vitro EMT model systems to identify a global EMT gene expression signature. By analyzing the commonly altered gene expression profiles we identified the transcription factor forkhead box protein F2 (Foxf2), neural cadherin (N-cadherin) and various cancer stem cell associated genes, to be upregulated during the EMT process. Our study revealed that Foxf2 upregulation, although not required to gain mesenchymal markers, is essential for the disruption of cell junctions and the downregulation of epithelial markers in NMuMG cells treated with TGFβ. We show that during EMT, Foxf2 regulates the expression of the E-cadherin mediators zinc finger E-box binding homeobox 1 and 2 (ZEB1 and ZEB2) and the inhibitor of differentiation 2 (Id2), leading to transcriptional repression of the CDH1 gene. Loss of E-cadherin results in the disruption of the polarity complex, a prerequisite for the dissociation and invasion of cancer cells. Furthermore, our investigations disclose that Foxf2 upregulation is required for TGFβ-mediated apoptosis in NMuMG cells by two major routes: direct transcriptional activation of the pro-apoptotic BH-3 only protein Noxa and negative regulation of EGFR-mediated survival signaling through the repression of its ligands Betacellulin and Amphiregulin. Substantiating the dual function of Foxf2 during EMT found by its implication in cell survival and cell motility, we show that high expression of Foxf2 correlates with good prognosis in early non-invasive stages of tumor development, but with poor prognosis in patients with advanced breast cancer. In contrast to Foxf2, our studies revealed that the expression of the well established mesenchymal marker N-cadherin is not required to induce EMT in NMuMG cells. Moreover we show that N-cadherin function is not essential to maintain the mesenchymal state and its depletion does not induce the reverse mesenchymal to epithelial transition. In addition, N-cadherin downregulation has no impact on the motility of mesenchymal cells nor on the neurite formation of pancreatic tumor cells. These data indicate that N-cadherin acts as a marker protein without evident function during the EMT process. In addition to our studies on Foxf2 and N-cadherin, we show that EMT leads to an increase in resistance to anoikis and the capacity to form mammospheres which is thought to correlate with cancer stemness and tumorigenicity. However, we could not detect a defined cancer stem cell subpopulation using cell surface markers nor a general increase of marker expression in mesenchymal cells. These results question whether the correlation of EMT and cancer stemness is intrinsic to certain systems or whether the commonly used surface markers are inadequate to generally detect cancer stem cells. In summary we identified the transcription factor Foxf2 as important regulator of EMT displaying a dual function in promoting apoptosis as well as invasive properties, while we were not able to detect cancer stemness or a specific function of N-cadherin during EMT. Thus, gaining more and better insights into the molecular function of Foxf2 may provide new strategies for anti-cancer treatments.